Ovarian tissue cryopreservation is one of the key approaches to fertility preservation. It does not require ovarian stimulation and can be done in a relatively short time and even in minors. While previous research indicated that primordial follicle losses are relatively small after freezing and thawing of ovarian tissue, a large fraction of the ovarian reserve is lost during the initial ischemia after ovarian transplantation. Thus any approach that can enhance and facilitate revascularization of ovarian transplants is likely to improve the outcome of the procedure. Hence, the long-term objective of this project is to develop novel strategies to improve tissue survival after transplantation of ovarian tissue in humans. The overarching hypothesis of our proposal is that the primordial follicle survival and ovarian graft function can be improved by pharmacological treatments enhancing vasculogenesis. This will be studied under the following specific aims: 1. To determine whether a ceramide-induced cell death pathway inhibitor, sphingosine-1 phosphate (S1P), protects against post-transplant ischemia-induced damage to ovarian primordial follicle pool in a human ovarian xenograft model. Recent studies have shown that S1P, an inhibitor of ceramide-induced apoptotic cell death, can enhance physiological vascularization processes. Thus we hypothesize that S1P will enhance primordial follicle survival during the initial ischemic phase after ovarian transplantation by promoting neovascularization of ovarian grafts. To test the effectiveness of S1P in augmenting ovarian transplant neovascularization, we will employ a human ovarian xenograft model based on Severe-Combined- Immunodeficiency (SCID) mice. We will determine whether S1P improves graft survival and neovascularization by primordial follicle survival, histological and molecular assessment of neovascularization, evidence of hypoxia by HIF-1a, as well as by utilization of intravital dye injections and innovative live imaging techniques. 2. To determine whether S1P treatment improves the functionality of frozen-thawed ovarian tissue transplants in a xenograft model. While S1P may enhance vascularization of ovarian transplants and increase survival of primordial follicles, this does not necessarily mean that these grafts will have extended function. Thus, we hypothesize that not only S1P will improve primordial follicle survival in ovarian xenografts but these follicles will be functional. A larger surviving functional primordial follicle pool should translate into extended functionality of ovarian grafts. To show that S1P treatment can thus enhance functionality of ovarian transplants, we will conduct long-term ovarian xenografting experiments. After 16 weeks of xenografting, functionality will be assessed and compared between S1P treated and control grafts by analysis of follicle and oocyte morphology, oocyte maturation and ovulation rates in response to hCG administration, steroid hormone production, as well as parthenogenic activation. PUBLIC HEALTH RELEVANCE: Premature ovarian failure and infertility are significant consequences of cancer treatments with major impact on quality of life and public health. Transplantation of cryopreserved ovarian tissue is one of the emerging key fertility preservation strategies in cancer patients but the technique is limited due to low survival of eggs in ovarian tissue. The overall goal of this project is to enhance ovarian graft survival and function by developing pharmacological treatments that can enhance new vessel development in ovarian transplants.